Because of these limitations in blood groups, forensic serologists had to look to different kinds of inherited biochemical markers to extend the discriminating power of typing. Fortunately, many of the systems of forensic interest turned out to be enzymes found on the red cell membrane. The main example is the enzyme phosphoglucomutase (PGM). PGM catalyzes the reversible conversion of glucose-1-phosphate and glucose-6-phosphate, with glucose-1.6-diphosphate as a co-factor.  PGM is an important metabolic enzyme and is found throughout the body.  It is expressed at many loci, and the form found in red cells is designated as the PGM 1 locus, usually written as PGM₁.  The PGM₁ locus is also expressed in semen, which increased its value in forensic serology.  There are two alleles, designated "1" and "2", giving the phenotypes PGM-1. PGM-2, and PGM 2-1.  Note that the locus is assumed, and the subscripted identifier has been omitted.  The population frequencies for the three phenotypes are approximately 59%, 36%, and 5%, respectively.  The actual frequencies vary by race and ethnicity.  Rare variants of the 1 and 2 alleles have been found.

Other red cell enzymes used in forensic biology include the following:

• erythrocyte acid phosphatase (EAP)
• esterase D (EsD)
• adenylate kinase (AK)
• adenosine deaminase (ADA)
• glyoxalase (GLO)

The enzymes vary in their stability in stains, the reliability of typing, the sensitivity of tests, and in their discriminating power.  Although discriminating power can be increased by testing for more than one enzyme, each individual test consumes sample, typically about six one-centimeter threads from a stain on cotton cloth.  One partial solution is to run more than one system at a time, and Multi Enzyme Systems (or MES) became popular for a time.  Typical combinations included PGM, EsD, and GLO, and PGM, ADA, and AK.

Identification of the polymorphisms in all the above systems depends on the same basic principles:

• The changes in structure affect the net charge of the isoenzymes.
• The isoenzymes can be separated by simple electrophoresis.
• The locations of the separated isoenzymes can be visualized by reactions that depend on the specific enzyme activity.

Starch gel was the usual separation medium, but cellulose acetate, polyacrylamide, and agarose were also used.  Most of the detection systems used a biochemical chain reaction in which the enzyme of interest reduced nicotinamide adenine dinucleotide phosphate (NADP) to NADPH with the concomitant conversion of MTT tetrazolium (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide) to the purple colored formazan in the presence of phenazine methosulfate (PMS). 

Some of the enzymes – EsD and EAP for example – can hydrolyze esters to produce a fluorescent compound that can be visualized under UV light.

A variant of electrophoresis is isoelectro focusing (IEF) where a pH gradient is formed during the electrophoresis and molecules move until the point in the gel at which they carry no charge.  IEF produces much sharper bands than slab gel electrophoresis.  Attempts to improve PGM separation by using IEF gave a surprising result, namely the discovery of a further two alleles, the expression of which was not detected by starch gel separations.  Each of the alleles detectable by starch gel electrophoresis had two alternate forms, designated as the "+" and "-" alleles.  Thus, the 10 phenotypes were comprised of the four homozygous forms PGM-1+, PGM-1-, PGM-2+, PGM-2-, and their heterozygous expressions.

Sometimes referred to as "PGM sub-typing," IEF was probably one of the best techniques available before the advent of DNA typing. The technique could type very low concentrations of enzyme and the enzyme itself was stable in blood and semen stains. A diagram showing the PGM sub-types is shown below.

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